Concentric Layers of Granule

The occurrence of concentric layers in granules was demonstrated using CLSM after staining by specific fluorochromes or FISH with specific oligonucleotide probes. The description of the layers is given in Table 6.2. Considering a microbial granule as a sphere with a diameter of 2.4 mm,

Table 6.2. Descriptions of layers in aerobically grown microbial granules grown in a column SBR with a medium containing ethanol or acetate

Table 6.2. Descriptions of layers in aerobically grown microbial granules grown in a column SBR with a medium containing ethanol or acetate

Layer

Average depth of layer from

Assumed function in

the surface of granule and

the granule

average thickness

Aerobic ammonia-oxidizing

70 ^m (depth); 30 ^m

It reflects the depth

bacteria

(thickness)

of oxygen

diffusion into

granule

Facultative anaerobic

Concentration increased to

Bacteria perform

enterobacteria

maximum at a depth of

both aerobic and

450 ^m and remained

anaerobic

stable at depths from

processes

450 to 850 ^m

Obligate anaerobic bacteria

850 ^m (depth); 150 ^m

It reflects the presence

Bacteroides spp.

(thickness)

anaerobic zone in

granule

Channels and pores by

Depth linearly depends on

Deeper diffusion of

penetration of 0.1 ^m

granule diameter by

nutrients

microspheres

equation (6.1)

Layer of active biomass

Thickness linearly depends

All bioactivities of the

on granule diameter

granule are

concentrated in this

layer

Polysaccharides

Low content to a depth of

It can decrease

500 ^m, reaching a

diffusion of nutrients

maximum at 650 ^m.

into granule through

Stable but low content at

the channels

depth from 800 to

1200 ^m

Core of dying cells in the

Depth was 1000 ^m.

Supply of monomers

center of granule

Diameter of this inner

and ammonia from

core depended on granule

this zone

diameter

the volumes of different zones can be calculated and compared with the experimental microbiological diversity of the granules (Table 6.3).

To determine the percentage of aerobic, facultative anaerobic, and anaerobic bacteria, cloning and sequencing of the 16S rRNA genes of the bacteria in the granules and phylogenetic analyses of the cloned sequences

Table 6.3. Average geometric and biological parameters of 2.4 mm spherical granule grown in a column SBR with a medium containing ethanol or acetate

Layer or zone in the

Geometric

Volume,

% of total

% of related

granule

parameters

mm3

volume of the granule

bacterial clones isolated from the granules

Aerobic

0.55 mm below

6.09

84.1

69 ± 7%

microorganisms

granule surface

in porous layer

Facultative

Between 0.55

0.97

13.4

9 ± 7%

anaerobic

and 0.85 mm

microorganisms

below granule surface

Obligate anaerobic

Between 0.85

0.15

2.0

2.1%

microorganisms

and 1.0 mm

(Bacteroides spp.)

Central core of dead

Depth is 1 mm.

0.03

0.5

-

and lyzed cells

Diameter is 0.4 mm

were performed (Tay et al., 2002b). Physiological property of the operational taxonomic units (OTUs), relation to oxygen, was inferred from the phylogenetic identification of OTUs. A CY5-labeled Ent1432 probe with the sequence 5/-CTTTTGCAACCCACT-3/ (Sghir et al., 2000) and with Tm of 45°C was used to detect enterobacteria. There is a statistically reliable correlation between the calculated volumes occupied by aerobic, facultative anaerobic, and anaerobic bacteria and the experimentally determined percentages of aerobic, facultative anaerobic, and anaerobic bacteria isolated from the granules.

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